​The bacterial flagellum is a fascinating molecular motor, present at the surface of many pathogenic bacteria, which allows bacterial motility through rotation. Currently we have only a low-resolution view for most parts of the flagellum. My project consists of using advances in single-particle Cryo-EM to get structural details at near atomic resolution, focused on the flagellar tip which is suspected to be involved in adhesion and surface recognition. With very few models of FliD capping protein in Campylobacter jejuni, my goal is to determine the natural oligomerisation state of the protein, observe its interaction with the rest of the flagellum and adhesion to various targets.

Antimicrobial resistance is at present one of the most challenging medical problems. Traditional antibiotics kill bacteria by targeting bacterial cell walls, a complex biological structure formed of cell membrane and peptidoglycan strands. Over time the bacteria develop strategies to counter the effects of drugs in order to maintain their cellular integrity and function. This project will focus on developing quantitative models rooted in equilibrium and non-equilibrium statistical mechanics and classical field theories applied to polymers, membranes, colloids, gels and other soft matter systems to predict the morphology and mechanical properties of growing bacterial cell walls based on their molecular structure and understand how this process is compromised by antibiotics. This theoretical project will be developed synergistically with imaging and biochemical studies (e.g. Atomic Force Microscopy) carried out in the groups of Prof. Simon Foster and Prof. Jamie Hobbs, focusing on the pathogen S. aureus (i.e. the hospital ‘super-bug’ MRSA).

My project aims to use imaging techniques to describe the molecular and mechanistic events of phagocytosis. We hope to develop mulitplex imaging methods to analyse changes in the physical properties of a cell, as well as its cell membrane and actin components during early stages of phagocytosis. To acheive this we will use advances in superresolution microscopy, fluorescence labelling, and computational software.

Staphylococcus aureus is a highly successful pathogen that can circumvent many aspects of immunity. Neutrophils, the most abundant of our white blood cells, are a critical defence against infection and yet S. aureus is able to render them ineffective. S. aureus does this by rupturing an intracellular neutrophil compartment known as the phagolysosome, which is where bacteria are normally contained and killed. Escape from the inhospitable phagolysosome allows S. aureus to replicate inside the neutrophil cytosol, eventually bursting the neutrophil open and becoming free to cause infection elsewhere. This project will use super-resolution and confocal microscopy techniques to determine how S. aureus disables the immune response in this way, observing interactions of S. aureus with subcellular components of human neutrophils on a molecular level, visualising early sub-cellular trafficking of the bacteria, interactions with endogenous microbicidal factors, phagosome lysis and escape on a molecular level. The ultimate aim of this project is to identify ways of preventing escape of S. aureus from the phagosome, in order to improve the host defence against this multi-drug resistant and life-threatening microorganism.

The bacterial cell wall is essential for viability. The major stress-bearing cell wall polymer that can withstand the internal cellular turgor pressure in Staphylococcus aureus is peptidoglycan (PG). Because of this, PG synthesis is the target of antibiotics such as penicillin.

The periplasmic space between the cell membrane and the cell wall is of key interest in this project, as it is where the final stages of PG synthesis occur and where penicillin acts. Our main objective is to understand how this periplasmic space is maintained when it would be expected that turgor would force the cytoplasmic membrane against the PG layer. This will further allow us to explore the effects of antibiotics on the maintenance of the bacterial periplasm.

Electron cryotomography (ECT) of frozen samples coupled with genetic and biochemical approaches will help us determine how the periplasm is established and maintained. Correlative microscopy using ECT and Stochastic Optical Reconstruction Microscopy (STORM) will be used to track changes in the cell membrane, PG position and, specifically, the periplasmic space during growth and the effects of antibiotics.

Microorganisms in the soil break down plant matter and recycle it back into the atmosphere as carbon dioxide. However, this process is limited by structures called microaggregates, ~100 µm clusters of tightly bound clay particles that cumulatively make a vast quantity of carbon biologically inaccessible. I am investigating how and why bacteria build houses as well as the combined effect of many clay houses on carbon sequestration. To this end we are using a wide diversity of tools, including molecular biology, microfluidic devices and physical modelling to understand how microscopic processes in the soil affect climate at the global scale.

Photodynamic (light-activated) therapy (PDT) is an emerging powerful approach to treat illnesses. It uses special drugs which are only toxic when illuminated by visible light, but remain non-toxic without light. This approach presents tremendous opportunities for targeted therapy and dramatically reduce side-effects. So far, the main focus of research in this area was on killing cells – especially cancer cells. This project aims at an even more ambitious target – we want to translate our recent developments in anti-cancer PDT to kill bacteria. Why is it such a challenge? Because bacteria invade the cells, and one must kill bacteria, but not the host cell. We will focus in particular on the bacterium Prophyromonas gingivalis, which is responsible for periodontal disease.It has been shown that when this bacterium invades host cells, it is resistant to both therapeutic agents and the immune response. We believe that one alternative to overcome resistance is antimicrobial photodynamic therapy (aPDT) which uses small-molecule light-activated drugs and targeted light applied directly to the gums to activate the drug.

Antibiotic resistant bacteria pose a considerable threat to human health, with a direct impact on morbidity, healthcare and the economy. MRSA is a well characterised human pathogen and is able to maintain virulence against many conventional penicillin’s. Surprisingly little is known about the exact method of penicillin-induced death in non-resistant strains. Using atomic force microscopy, this work is intended to elucidate the mechanical properties of Staphylococcus aureus prior to, and after the addition of antibiotics, to better understand the life and death of the bacterium.

Context: Most antibiotics target the peptidoglycan polymer of actively growing bacteria and are thought to inhibit the growth and division process, disrupting the cell wall and ultimately leading to lysis and the death of the cell. We now know that this is an overly simplistic view and that these actions alone cannot accurately account for the demise of the bacterium. This work is designed to better understand the mechanical nature of peptidoglycan and to characterise the effects of antibiotics on its integrity and growth.

The human pathogen Methicillin-resistant Staphylococcus aureus (MRSA) is a principal cause of difficult-to-treat infections worldwide, as it has developed resistance to β-lactam antibiotics. Resistance is due to of Penicillin Binding Protein 2A (PBP2A) encoded by the mecA gene. PBP2A has a low affinity for β-lactams and is able to synthesize essential cell wall peptidoglycan (PG) in their presence. However how PBP2A interacts with the rest of the cell division machinery and mediates PG biosynthesis is unknown. I will take a combination of genetic and super resolution microscopy approaches to investigate how such an important pathogen is able to evade killing by some of the most important antibiotics ever developed.

DNA replication and inheritance is an important process required for all life forms, our group focuses on the DNA inheritance specifically in the bacteria Vibrio cholerae. V.cholerae have 2 chromosomes, both requiring their own chromosome segregation system. In vivo studies have shown the patterns of chromosome segregation to be distinctly different to one another. The larger chromosome 1 segregates asymmetrically with the origin of replication moving from one pole to another, while the origin of chromosome 2 moves from the half-cell to the quarter-cell position. However, the molecular mechanism of action of chromosome segregation for both chromosomes is yet to elucidated. My research involves uncovering the molecular details of V.cholerae ’s chromosome 1 segregation, using Total Internal Reflection Fluorescence (TIRF) Microscopy.

Optical microscopy has played a significant role in biological research due to its non-contact, minimal invasive nature enabling in vivo investigation. The well-known Abbe diffraction limit has initiated the development of super resolution optical microscopy which opens tremendous scope for the study of biological samples. This project entails developing a novel illumination system for super resolution imaging in association with an optics company Cairn-Research. The system is based on Digital Light Processing (DLP) which can be used to generate patterns of illumination and/or detection. The system allows us to collect in focus (conjugate image) and out of focus (non-conjugate image) light coming from specimen in two separate cameras making it a dual path Programmable Array Microscope (PAM) which can result in significantly low noise and sharper focus. We can achieve spatial and temporal patterning of illumination by controlling the Digital Micro-mirror Device (DMD) pixel by pixel. The major advantage of this system is the incorporation of various well established imaging techniques such as confocal or Structured Illumination Microscopy (SIM) into one single setup. This versatile system is to be developed to address specific problems in biomedical science enabling the user to change the imaging technique depending on the sample in study.

Stochastic optical reconstruction microscopy (STORM) is a super resolution imaging technique that offers a lateral resolution of around ~20nm, yet cannot achieve a comparable axial resolution for 3D imaging. The goal of my project is to develop and build a new 3D-STORM microscope which will combine the super critical angle technique with the ability to perform 3-color imaging. It is hoped the system will allow us to obtain 3D multi-color images with an isotropic resolution of ~10nm. The method will be applied to image centriolar proteins, in order to create a quantitative nanoscale atlas of the centrosome throughout the cell cycle.

During the last few years super resolution microscopy techniques have begun to revolutionize our understanding of the organization of bacterial cells. The goal of the project is to study bactofilins, important morphogenic proteins that are part of the newly discovered bacterial cytoskeleton (see Mol Microbiol 2011, 80:1031-101). This project will use a suite of super resolution light and electron microscopy to study the structure, function and dynamics of these important proteins.

Flap endonucleases (FENs) play a vital role in DNA replication, repair and recombination in all living cells. Aspects of how FENs locate their branched DNA substrates and the conformational changes associated with DNA hydrolysis remain unclear. In my project, we are trying to address these questions using a combination of direct atomic force microscopy (AFM) imaging with conventional biochemical and structural approaches. Development of imaging of DNA to major and minor groove resolution using approaches initially proven for membrane protein imaging, has provided the potential to directly visualize local strain within a single molecule during an interaction with a bound protein. Using AFM we aim to explore how FENs engage their substrate using imaging of DNA-FEN complexes to characterize their hydrolysis.